Metabolic cage system

At weeks 5 and 12, animals were placed into metabolic cages (Tecniplast Metabolic Cage System, Buguggiate, Italy) for 24 h to measure urine creatinine and protein concentrations by standard laboratory methods as described previously^{35 (link)–37 (link),52 (link)} (Fig. 1). Blood was collected from the saphenous vein at week 4 and from the abdominal aorta at week 13 to measure serum carbamide (urea), creatinine, and lipid levels to verify the development of CKD^{35 (link)–37 (link),52 (link)}. Serum urea and creatinine levels were quantified by kinetic UV spectrophotometric method using urease and glutamate dehydrogenase enzymes and Jaffe’s method, respectively, using the Roche Diagnostics reagents and platform analyzers (Hoffmann-La Roche Ltd., Basel, Switzerland)^{35 (link),52 (link)}. At week 5 and the endpoint, urine creatinine and volume and serum creatinine concentration were measured^{35 (link),52 (link)}. Creatinine clearance, a renal functional indicator, was calculated using the standard formula (urine creatinine concentration [μM] × urine volume for 24 h [mL])/(serum creatinine concentration [μM] × 24 × 60 min)^{40 (link),52 (link)}. At weeks 5 and 13, total serum cholesterol and triglyceride levels were measured by Roche Cobas 8000 analyzer system using enzymatic colorimetric assays (Roche, Hoffmann-La Roche Ltd., Basel, Switzerland)^{36 (link)}.

At weeks 5 and 12, animals were placed into metabolic cages (Tecniplast Metabolic Cage System, Buguggiate, Italy) for 24 h to measure urine creatinine and protein levels. As described previously, urine creatinine and protein levels were measured by standard laboratory methods [34 , 36 (link), 39 (link), 40 (link)]. Blood was collected from the saphenous vein at week 5 and from the abdominal aorta at week 13 to measure serum carbamide (urea) and creatinine levels to verify the development of CKD. Urea and creatinine levels in serum were quantified by kinetic UV spectrophotometric method using urease and glutamate dehydrogenase enzymes according to the Jaffe method. The reagents and the platform analyzers were from Roche Diagnostics (Hoffmann-La Roche Ltd., Basel, Switzerland) [39 (link), 40 (link)]. Creatinine clearance, an indicator of renal function, was calculated according to the standard formula (urine creatinine concentration [μM] × urine volume for 24 h [mL])/(serum creatinine concentration [μM] × 24 × 60 min). At weeks 5 and 12 or 13, urine creatinine and volume and serum creatinine concentration were measured [39 (link), 40 (link)].